It is now clear that monoglutamyl folates, upon entering mammalian cells, are rapidly transformed to polyglutamyl derivatives, that these compounds function as substrates for the folate dependent enzymes and that mutational loss of the enzyme(s) that synthesizes polyglutamyl folates is a lethal deletion. It is also clear that methotrexate and related folate analogs are substrates for this enzyme and that antifol polyglutamates may be involved in the selective toxicity of the antifols. The overall goals of this work are to evaluate folate polyglutamate synthetase as a target enzyme for cancer chemotherapy and to study the relationship between the formation of antifol polyglutamates and the chemotherapeutic utility of these drugs. The proposed work would (1) purify and characterize folate polyglutamate synthetase from normal and malignant mammalian tissue, (2) define the structure-activity relationships for substrate and inhibitor activity for these enzymes, (3) investigate the relationship between tissue differentiation and enzyme activity, (4) study the chemotherapeutic utility of the formation of polyglutamates of various antifols, and (5) define the biochemical effects of inhibitors of folate polyglutamate synthetase. This project offers a novel and rationally conceived class of folate antimetabolites which differ from the classical antifols typified by methotrexate in the site of induced inhibition of folate metabolism.